1. Technical Field
This invention generally relates to analyte sensors and methods for detecting or quantifying analytes. More particularly, this invention relates to fluorescent protein sensors for detecting and quantifying analytes, including Ca2+ and Tb3+, or for detecting proteins under in vivo and in vitro conditions.
2. Prior Art
Analytes, including Ca2+, are essential to life and control numerous cellular processes such as cell division and growth, secretion, ion transport, muscle contraction, and neuron signaling through interaction with proteins. Further, analytes such as calcium, magnesium, iron and other metal ions are essential to biological systems through interaction with nucleic acid, lipids, carbohydrates and biometabolic molecules. Not only are many analytes essential structural components, e.g. Ca2+ in teeth and bones, but analytes also act as second messengers regulating many biological processes during the birth, life, and death of cells. Furthermore, analyte-mobilizing agents such as ATP, histamine, glutamine, and second messengers such as inositol triphosphate (IP3) and CADPR affect the cytosolic concentration of Ca2+ with defined spatio-temporal patterns.
As temporal and spatial changes in analyte concentration have significant consequences in biological processes, detection and quantification of the local analyte concentration in vitro or in vivo may provide insight into physiological processes and a number of human diseases. For example, it is known that changes in Ca2+ concentration have a role in neuronal signaling, muscle contraction, and cell development and proliferation. Further, cellular processes such as gene expression, protein folding, metabolism and synthesis are controlled by different levels and kinetic properties of analyte signaling. Additionally, as diseases such as Alzheimer's disease, cancer, and lens cataract formation are known to be associated with altered Ca2+ signaling, improved quantification and detection of such signals may provide valuable insight into the aforementioned diseases. Thus, detecting and quantifying changes in analytes that occur in cells or organisms may provide important insight into biological activities and diseases.
Specifically, for illustrative purposes, Ca2+ binds many molecules, especially proteins, at different environments to regulate their functions. Currently more than 1000 calcium binding proteins are known in every kingdom, from mammalian to plants to bacteria. For example, calcium binds to calmodulin to trigger this protein to regulate over 100 processes in almost every compartment of the cell. Many calcium sensor receptors, growth factors, and cell adhesion molecules are directly regulated by calcium binding. Ca2+ signal changes are used as one of the best ways to monitor neuron science, brain and behaviors. Therefore, accurate measurement of Ca2+ concentration in a broad concentration range under in vitro or in vivo (both intracellular and extracellular) conditions by non-invasive techniques, without significantly disrupting cellular functions, is of paramount importance. As such, the constant Ca2+ homeostasis results in local Ca2+ variations.
Accordingly, there is always a need for an improved analyte sensor for quantifying and detecting analyte concentrations and changes thereof in both in vivo and in vitro systems and for probing the functionality of analyte binders and for methods of constructing and engineering new binding sites. Due to the importance of analytes in the physiology of biological and cellular processes, it is essential to develop analyte binding sites for use in proteins, e.g. fluorescent protein, and methods constructing such binding sites. Further, it is important to develop an analyte sensor that can detect changes of the analyte concentration in the microenvironment inside or outside of cells in real time. Such sensors, which can detect changes in microenvironments, are useful as probes of cellular events involving changes in such microenvironments due to movement of molecules in solution or the special location of molecules associated with cell membranes. It is to these needs among others that the present invention is directed.